LOCUS Exported 6499 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses 3xFLAG-GABARAPL1-EGFP in mammalian cells, for monitoring ATG4 activity towards GABARAPL1. ACCESSION . VERSION . KEYWORDS pDEST-CMV 3xFLAG-GABARAPL1-GFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6499) AUTHORS Agrotis A, Pengo N, Burden JJ, Ketteler R TITLE Redundancy of human ATG4 protease isoforms in autophagy and LC3/GABARAP processing revealed in cells. JOURNAL Autophagy. 2019 Jan 21. doi: 10.1080/15548627.2019.1569925. PUBMED 30661429 REFERENCE 2 (bases 1 to 6499) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6499 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(48..67) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 239..618 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 619..822 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 773..793 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" CDS 859..924 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" protein_bind 928..952 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" protein_bind complement(1307..1331) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" CDS 1348..2067 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 1348..2064 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(1393..1414) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1654..1673) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2001..2022 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(2094..2111) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2100..2324 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 2370..2798 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2457..2476) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2667..2688 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(2807..2827) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 2812..3141 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 2992..3127 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3054..3073 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3208..4002 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(3262..3281) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 3872..3891 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4176..4297 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4213..4232) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4267..4286 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4346..4362) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4346..4362) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4359..4381) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4370..4386 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(4394..4424) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4439..4460 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4577..4594) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4748..5336) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4828..4847) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5507..6367) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 6130..6149 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6368..6472) /gene="bla" /label=AmpR promoter ORIGIN 1 cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct 61 gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag 121 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat 181 ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga 241 cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca 301 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac 361 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact 421 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa 481 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg 541 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta 601 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg 661 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg 721 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg 781 ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctcgtttagt gaaccgtcag 841 aattgatctg gtaccatgga ctacaaagac catgacggtg attataaaga tcatgacatc 901 gattacaagg atgacgatga caagcttaca agtttgtaca aaaaagcagg cttaatgaag 961 ttccagtaca aggaggacca tccctttgag tatcggaaaa aggaaggaga aaagatccgg 1021 aagaaatatc cggacagggt ccccgtgatt gtagagaagg ctccaaaagc cagggtgcct 1081 gatctggaca agaggaagta cctagtgccc tctgacctta ctgttggcca gttctacttc 1141 ttaatccgga agagaatcca cctgagacct gaggacgcct tattcttctt tgtcaacaac 1201 accatccctc ccaccagtgc taccatgggc caactgtatg aggacaatca tgaggaagac 1261 tattttctgt atgtggccta cagtgatgag agtgtctatg ggaaataccc agctttcttg 1321 tacaaagtgg ttcgatctag actcgagatg gtgagcaagg gcgaggagct gttcaccggg 1381 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc 1441 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc 1501 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc 1561 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa 1621 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc 1681 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc 1741 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc 1801 tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac 1861 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac 1921 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac 1981 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact 2041 ctcggcatgg acgagctgta caagtaatta attaagataa acccgctgat cagcctcgac 2101 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct 2161 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct 2221 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg 2281 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag 2341 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc 2401 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc 2461 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa 2521 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact 2581 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt 2641 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa 2701 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt 2761 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag 2821 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc 2881 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa 2941 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc 3001 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat 3061 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt 3121 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat 3181 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct 3241 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc 3301 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc 3361 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc 3421 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg 3481 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag 3541 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc 3601 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt 3661 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc 3721 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc 3781 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg 3841 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag 3901 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg 3961 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg 4021 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct 4081 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc 4141 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg 4201 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt 4261 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct 4321 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc 4381 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 4441 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 4501 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 4561 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 4621 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 4681 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 4741 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 4801 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 4861 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 4921 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 4981 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 5041 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 5101 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 5161 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc 5221 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 5281 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 5341 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 5401 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 5461 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 5521 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 5581 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 5641 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 5701 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 5761 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 5821 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 5881 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 5941 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 6001 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 6061 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 6121 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 6181 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 6241 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 6301 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 6361 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 6421 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 6481 cccgaaaagt gccacctga //